Bacterial cytokinesis is mediated by the septal ring (SR), a cytoskeletal-like organelle that forms at the future site of cell fission and then drives te coordinate invagination of the inner membrane, peptidoglycan, and outer membrane layers during the process. The long-term goals of this project are to understand at a molecular level: i) The composition and architecture of the SR, ii) How the SR assembles from its different components, iii) How the proper site for its assembly is determined, iv) How the SR drives cell fission, and v) How SR function is coordinated with other cell cycle events. In E.coli, the SR consists of at least ten essential division proteins and over twenty other proteins with redundant and/or non-essential activities in the division process. SR development involves assembly of an early Z-ring (ZR) intermediate, recruitment of additional proteins to form a fission-competent SR, and further modifications after the onset of the fission process. The ZR consists of polymers of the tubulin-like GTPase FtsZ that are directly decorated by at least four other division proteins. To understand how FtsZ polymers form a membrane-associated ring, it remains an important challenge to reconstitute ZRs in vitro with native proteins. We found conditions wherein purified native FtsZ together with two other ZR proteins can form ZR-like structures in lipid vesicles. We will optimize the system to further study the properties of these structures, and to define the requirements for assembly and possible activities of such structures in vitro. In parallel, we will better define the requirements for ZR assembly in vivo. FtsN is the last essential protein to join the SR and our data indicates it contains a small periplasmic peptide domain that starts a positive feedback loop to initiate and/or sustain the constriction process. DedD is an FtsN- like SR protein with related but distinct properties and is essential for cells with limiting FtsN activty. We will identify the relevant critical SR partners of FtsN and DedD and characterize the role of the interactions in stimulating the constriction process. The Tol-Pal proteins form a trans envelope complex that is well conserved in Gram negative bacteria. We showed that it is recruited to the SR in constricting cells and required for coordinated invagination of the outer membrane with the other envelope layers. We propose that Tol-Pal does so by establishing a wave of transient (trans) envelope connections that trail the SR as it constricts the inner membrane and forms the two new polar peptidoglycan caps. We will test important aspects of the model by in vivo and in vitro experiments, determine how the complex is recruited to the SR, and further define the specific division defects in cells lacking Tol-Pal and/or other proteins tha affect outer membrane invagination. A number of SR proteins are, or are actively pursued as, drug targets. This project may lead to the identification of additional promising targets for, and rational designs of, new anti-bacterial drugs.